Oncolytic virus (OV) is an emerging anti-cancer agent that induces cancer selective cytotoxicity and anti- cancer immune response, and is now approved for advanced melanoma. However, extracellular matrix (ECM) within the tumor microenvironment poses a challenge in OV delivery. Hyaluronic acid (HA) in the brain ECM presents a barrier to OV, limiting its distribution within the tumor. We have shown that HA is expressed in abundance in a panel of orthotopic brain tumor glioblastoma (GBM) xenografts generated with patient-derived GBM cancer stem cells (GSC) lines, and that an oncolytic adenovirus expressing hyaluronidase, ICOVIR17, degrades HA, and mediates increased intratumoral spread and anti-GBM efficacy in orthotopic GBM models. Recently, we have reported that mesenchymal stem cells (MSC) serve as a robust cell carrier of OV and efficiently deliver OV to GBM. Furthermore, encapsulation of ICOVIR17- loaded MSC in HA-based biodegradable hydrogel and its application into tumor resection cavities efficiently delivered virus to tumor remnants and greatly extended survival of brain tumor bearing animals. These findings highlight the translational potential of MSC delivery of ICOVIR17 as a novel post-operative therapeutic strategy for GBM. However, two critical questions remain unanswered: 1) how to treat invasive GBM tumors in the brain with MSC/ICOVIR17; and 2) whether or how host immune system impacts the efficacy of MSC/ICOVIR17. As Aim 1, we will determine the therapeutic efficacy of MSC/ICOVIR17 therapy in PDX models of invasive GBM. By using patient-derived GBM xenograft (PDX) models that exhibit extensive invasiveness in the brain, we will test 2 routes of MSC/ICOVIR17 delivery: implantation in resection cavities and intracarotid arterial delivery. The fate of tumor cells, MSC, and ICOVIR17 will be systematically analyzed by optical imaging and correlated histopathological studies. The therapeutic efficacy of MSC/ICOVIR17 will be determined by survival analysis. In immune-competent mice, we have found that GBM resection elicits CD4+ dominant T cell recruitment to the tumor bed in the brain. We hypothesize that ICOVIR17 degradation of HA within GBM would increase OV spread and further recruit immune cell infiltration to the brain tumor, and combining the MSC/ICOVIR17 strategy with immune checkpoint inhibition would cooperate to boost efficacy in syngeneic GBM models in immunocompetent mice. As Aim 2, we will determine the therapeutic efficacy of locally delivered MSC/ICOVIR17 in immunocompetent GBM models as monotherapy and as combination with immune checkpoint inhibitors. We will assess the therapeutic efficacy and cellular immune responses after intracavitary implantation of MSC/ICOVIR17 in 005 and CT-02A GBM models, and test combination therapy with anti-PD-1 for animal survival effects. These studies will provide data that are vital for rationally designing a GBM clinical trial testing MSC/ICOVIR17.